Structural requirements for binding of adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) to human neutrophils.
Human polymorphonuclear leukocytes (PMN) respond via pertussis toxin-sensitive pathways to extracellular nucleotides with an elevation in intracellular calcium ([Ca2+]i) and enhancement of the O2- generation induced by the chemotactic peptide N'-formyl-Met-Leu-Phe (fMLP). Binding studies with adenosine 5'-O-(3-thio[35S]triphosphate) ([35S]ATP gamma S) have recently identified at least two classes of receptors on intact human neutrophils. In this study, we further characterize nucleotide binding to this receptor with respect to its specific structural requirements. Utilizing [35S]ATP gamma S as the primary ligand and various nucleotides and ATP analogues, competitive binding studies demonstrate that: (1) the triphosphate tail is essential for maximal receptor binding; (2) chemical modifications of the phosphate tail have profound effects on binding efficacy; (3) the base ring is recognized by the receptor, with purines being preferentially recognized; and (4) except for a spacing function, the ribose ring of nucleotides does not appear to be important for nucleotide binding. In addition, we demonstrate that the presence of divalent cations inhibits [35S]ATP gamma S binding, suggesting that the tetraanionic form of ATP (ATP4-) is the nucleotide species reactive with the receptor.
Yu
G H
GH
Department of Pathology, University of Michigan Medical School, Ann Arbor 48109.
Ward
P A
PA
D000227:Adenine Nucleotides / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008274:Magnesium / Q000494:pharmacology; D015394:Molecular Structure; D009504:Neutrophils / Q000378:metabolism*; D011983:Receptors, Purinergic / Q000378:metabolism
D000227:Adenine Nucleotides; D011983:Receptors, Purinergic; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D008274:Magnesium; D002118:Calcium